Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.
In this paper, we present a combined theoretical and experimental study of the propagation of calcium signals in multicellular structures composed of human endothelial cells. We consider multicellular structures composed of a single chain of cells as well as a chain of cells with a side branch, namely a “T” structure. In the experiments, we investigate the result of applying mechano-stimulation to induce signaling in the form of calcium waves along the chain and the effect of single and dual stimulation of the multicellular structure. The experimental results provide evidence of an effect of architecture on the propagation of calcium waves. Simulations based on a model of calcium-induced calcium release and cell-to-cell diffusion through gap junctions shows that the propagation of calcium waves is dependent upon the competition between intracellular calcium regulation and architecture-dependent intercellular diffusion.
Calcium wave signal has been found in a wide variety of cell types. Over the last years, a large number of calcium experiments have shown that calcium signal is not only an intracellular regulator but is also able to be transmitted to surrounding cells as intercellular signal. This paper focuses on the development of an approach with complementary integration of theoretical and experimental methods for studying the multi-level interactions in multicellular architectures and their effect on collective cell dynamic behavior. We describe new types of higher-order (across structure) behaviors arising from lower-order (within cells) phenomena, and make predictions concerning the mechanisms underlying the dynamics of multicellular biological systems. The theoretical approach describes numerically the dynamics of non-linear behavior of calcium-based signaling in model networks of cells. Microengineered, geometrically constrained networks of human umbilical vein endothelial cells (HUVEC) serve as platforms to arbitrate the theoretical predictions in terms of the effect of network topology on the spatiotemporal characteristics of emerging calcium signals.
Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. The standard culture-based diagnosis of UTI has a typical delay of two to three days. In the absence of definitive microbiological diagnosis at the point of care, physicians frequently initiate empirical broad-spectrum antibiotic treatment, which has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Similar to how blood glucose sensors revolutionized the management of diabetes and pregnancy tests are now conducted at home, biosensors are poised to significantly improve UTI diagnosis. Biosensors are amenable to integration with microfluidic technology for point-of-care applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing and hurdles in the translation of biosensor technology from bench to bedside.
Cellular processes are regulated by various mechanical and physical factors in their local microenvironment such as geometric confinements, cell-substrate interactions, and cell-cell contact. Systematic elucidation of these regulatory mechanisms is crucial for fundamental understanding of cell biology and for rational design of biomedical devices and regenerative medicine. Here, we report a generally applicable plasma lithography technique, which performs selective surface functionalization on large substrate areas, for achieving long-term, stable confinements with length scales from 100 nm to 1 cm toward the investigation of cell-microenvironment interactions. In particular, we applied plasma lithography for cellular confinement of neuroblastomas, myoblasts, endothelial cells, and mammary gland epithelial cells, and examined the motion of mouse embryonic fibroblasts in directionality-confined environments for studying the effect of confinements on migratory behavior. In conjunction with live cell imaging, the distance traveled, velocity, and angular motion of individual cells and collective cell migration behaviors were measured in confined environments with dimensions comparable to a cell. A critical length scale that a cell could conceivably occupy and migrate to was also identified by investigating the behaviors of cells using confined environments with subcellular length scales.
Electrothermal flow is a promising technique in microfluidic manipulation toward laboratory automation applications, such as clinical diagnostics and high throughput drug screening. Despite the potential of electrothermal flow in biomedical applications, relative little is known about electrothermal manipulation of highly conductive samples, such as physiological fluids and buffer solutions. In this study, the characteristics and challenges of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. Electrothermal flow was shown to create fluid motion for samples with a wide range of conductivity when the driving frequency was above 100 kHz. For samples with low conductivities (below 1 S/m), the characteristics of the electrothermal fluid motions were in quantitative agreement with the theory. For samples with high conductivities (above 1 S/m), the fluid motion appeared to deviate from the model as a result of potential electrochemical reactions and other electrothermal effects. These effects should be taken into consideration for electrothermal manipulation of biological samples with high conductivities. This study will provide insights in designing microfluidic devices for electrokinetic manipulation of biological samples toward laboratory automation applications in the future.
Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.
The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).
We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.
Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune-response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but more informative than the current standard.
Electrochemical biosensor; Amperometry; Urinary diagnostics; Urinary tract infections; Biomarkers
Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.
The next generation of tissue engineered constructs (TECs) requires the incorporation of a controllable and optimized microstructure if they are to chemically, mechanically, and biologically mimic tissue function. In order to obtain TECs with optimized microstructures, a combination of spatiotemporally regulated mechanical and biochemical stimuli is necessary during the formation of the construct. While numerous efforts have been made to create functional tissue constructs, there are few techniques available to stimulate TECs in a localized manner. We herein describe the design of a microdevice which can stimulate TECs in a localized, inhomogeneous, and predefined anisotropic fashion using ferromagnetically doped polydimethylsiloxane microflaps (MFs). Specifically, a sequential magneto-structural finite element model of the proposed microdevice is constructed and utilized to understand how changes in magnetic and geometrical properties of the device affect MF deflection. Our study indicates that a relatively small density of ferromagnetic material is required to result in adequate force and MF defection (175 μm ~7% TEC strain). We also demonstrate that MF to magnet distance is more important than inherent MF magnetic permeability in determining resulting MF deflection. An experimental validation test setup was used to validate the computational solutions. The comparison shows reasonable agreement indicating a 5.9% difference between experimentally measured and computationally predicted MF displacement. Correspondingly, an apparatus with two MFs and two magnets has been made and is currently undergoing construct testing. The current study presents the design of a novel magnetic microactuator for tissue engineering applications. The computational results reported here will form the foundation in the design and optimization of a functional microdevice with multiple MFs and magnets capable of stimulating TECs in nonhomogenous and preferred directions with relevant spatial resolution.
Tissue engineering; Biomechanics; Magnetic; Microactuator; Finite element
This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic E. coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 micrometer or less in depth, the effective oxygenation can sustain the growth of E. coli to over 109 cfu/ml without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 hours compared to days in standard clinical procedures facilitating diagnostics at the point of care.
Cartridge-based microfluidics is a promising technology for clinical diagnostics. By miniaturizing the fluid-handling processes required for genomic and proteomic analyses, reagent and specimen volume is minimized along with the size of the system. We demonstrate an automated microfluidic system capable of performing six multiplexed genomic and proteomic analyses simultaneously, by means of an integrated electrochemical sensor and embedded controls.
microfluidics; electrochemical sensor; multiplexed assay; quantitative; molecular analysis; point of care; clinical diagnostics
In response to oxidative stress, the transcription factor NF-E2-related factor 2 (Nrf2) controls the fate of cells through transcriptional upregulation of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis. As a result, activation of the Nrf2 pathway, by naturally-occurring compounds or synthetic chemicals at sub-toxic doses, confers protection against subsequent toxic/carcinogenic exposure. Thus, the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention. Interestingly, recent emerging data has revealed the “dark” side of Nrf2. Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy. This review is primarily focused on the role of Nrf2 in cancer, with emphasis on the recent findings indicating the cancer promoting function of Nrf2 and its role in acquired chemoresistance.
Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers. Here, we report that inhibition of NF-E2-related factor 2 (Nrf2) may be a promising strategy to combat chemoresistance. Nrf2 is a critical transcription factor regulating a cellular protective response that defends cells against toxic insults from a broad spectrum of chemicals. Under normal conditions, the low constitutive amount of Nrf2 protein is maintained by the Kelch-like ECH-associated protein1 (Keap1)-mediated ubiquitination and proteasomal degradation system. Upon activation, this Keap1-dependent Nrf2 degradation mechanism is quickly inactivated, resulting in accumulation and activation of the antioxidant response element (ARE)-dependent cytoprotective genes. Since its discovery, Nrf2 has been viewed as a ‘good’ transcription factor that protects us from many diseases. In this study, we demonstrate the dark side of Nrf2: stable overexpression of Nrf2 resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin and etoposide. Inversely, downregulation of the Nrf2-dependent response by overexpression of Keap1 or transient transfection of Nrf2–small interfering RNA (siRNA) rendered cancer cells more susceptible to these drugs. Upregulation of Nrf2 by the small chemical tert-butylhydroquinone (tBHQ) also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, we provide evidence that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not limited to certain cancer types or anticancer drugs and thus can be applied during the course of chemotherapy to treat many cancer types.
Groundwater contaminated with arsenic imposes a big challenge to human health worldwide. Using natural compounds to subvert the detrimental effects of arsenic represents an attractive strategy. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of the cellular antioxidant response and xenobiotic metabolism. Recently, activation of the Nrf2 signaling pathway has been reported to confer protection against arsenic-induced toxicity in a cell culture model.
The goal of the present work was to identify a potent Nrf2 activator from plants as a chemopreventive compound and to demonstrate the efficacy of the compound in battling arsenic-induced toxicity.
Oridonin activated the Nrf2 signaling pathway at a low subtoxic dose and was able to stabilize Nrf2 by blocking Nrf2 ubiquitination and degradation, leading to accumulation of the Nrf2 protein and activation of the Nrf2-dependent cytoprotective response. Pretreatment of UROtsa cells with 1.4 μM oridonin significantly enhanced the cellular redox capacity, reduced formation of reactive oxygen species (ROS), and improved cell survival after arsenic challenge.
We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated. Furthermore, we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.
antioxidant responsive element; antitumor; ARE; arsenic; chemoprevention; diterpenoid; Keap1; Nrf2; oridonin; oxidative stress; rubescensin
Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations. In particular, trimethoprim and gentamicin were identified to work synergistically for inhibiting the bacterial growth. Sensitivity analysis indicated gentamicin functions as a synergist for trimethoprim, and reduces its minimum inhibitory concentration for 40-fold. Validation study also confirmed that the trimethoprim-gentamicin synergistic cocktail effectively inhibited the growths of multiple strains of uropathogenic clinical isolates. With its effectiveness and simplicity, the MACO scheme possesses the potential to serve as a generic platform for identifying synergistic antimicrobial cocktails toward management of bacterial infection in the future.
The transcription factor Nrf2 has emerged as a master regulator for the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), two well-studied inducers for neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 (NQO1) in a dose- and time- dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M (NF-M) and microtubule-associated protein 2 (MAP-2). Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to that from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases.
Nrf2; Keap1; Oxidative Stress; Neuronal differentiation; SH-SY5Y; NQO1